1. Field of the Invention
The present invention relates to fields of protein biology and diagnostics. More particularly, the present invention relates to improved muteins of streptavidin that specifically yet reversibly bind biotin and strepatavidin binding peptide tagged proteins.
2. Description of Related Art
Wild-type streptavidin is a tetrameric protein with four identical subunits. Two dimers self-associate to form a tetrameric structure. Each subunit can bind one biotin tightly with a dissociation constant (Kd) in the range of 10−13 to 10−14 M (Wilchek and Bayer, 1990). This binding is considered to be irreversible and streptavidin has been applied to capture and immobilize biotinylated biomolecules. It is widely used in development of many diagnostic kits, biosensor chip, protein and DNA arrays and Western blot studies. However, wild-type streptavidin is not suitable for purification of biotinylated biomolecules. To extend its application, it would be ideal to develop engineered streptavidin muteins with reversible biotin binding ability so that these muteins can be applied to purify biotinylated molecules, to study protein-protein interactions (with one of the interacting proteins to be biotinylated) and to develop reusable biosensor chips and bioreactors. Traditional bioreactors will have enzymes chemically immobilized. After many rounds of usage, bioreactors with the immobilized enzymes will become useless when the immobilized enzymes lose their activities. With an engineered streptavidin that can bind biotin in a reversible manner, one can immobilize these engineered streptavidin proteins to the bioreactor. The enzymes of interest can then be biotinylated and loaded to bioreactors with the immobilized streptavidin muteins to generate functional bioreactors. When the enzymes lose their activity, these inactive enzymes can be eluted off by biotin and the bioreactor can be reloaded with a new batch of biotinylated enzymes.
To develop streptavidin muteins with reversible binding ability, two approaches are common. The first approach is to replace one or more streptavidin amino acid residues that are critical in biotin binding with different residues. These changes can result in lowering the biotin binding affinity in these muteins (Qureshi et al., 2001; U.S. Pat. No. 6,312,916 B1). The second approach is to develop recombinant monomeric streptavidin (Wu and Wong, 2005a). This is based on the fact that a streptavidin subunit does not have a complete biotin binding pocket. A biotin binding pocket in subunit A requires a tryptophan 120 (Trp-120) residue from subunit D. This Trp-120 has been demonstrated to play an important role in biotin binding (Chilkoti et al., 1995).
While purification of biotinylated biomolecules using monomeric avidin offers one of the cleanest approaches, the biotinylation of molecules either chemically or enzymatically requires steps that can be time consuming, labor intensive and costly. To overcome this problem, several streptavidin binding peptide tags have been developed. One of them is the 38-amino-acid streptavidin binding peptide (SBP) tag (Keefe et al., 2001) that can bind streptavidin with high affinity (Kd˜2.5 nM) without biotinylation. It works well whether the tag is at the N-terminal, internal or C-terminal position of the recombinant protein (Van Leene et al., 2008; Kobayashi et al., 2008). Biotin added to the elution buffer acts as an effective competitor to elute the bound SBP-tagged protein off. However, as streptavidin binds biotin tightly (Kd˜10−14 M) (Green, 1990), the streptavidin matrix essentially can only be used once, and this drawback makes the purification very costly. In order to vitalize this powerful purification technology, it would be ideal to have an engineered streptavidin that not only can bind biotin reversibly, but one that retains a high SBP tag binding strength. The biotin binding strength (Kd) of this engineered streptavidin should be ˜10−8 M. Thus, biotin should be strong enough to displace the SBP tag from streptavidin, but not bind irreversibly to streptavidin. Consequently, the affinity matrix could be regenerated by a simple washing step and can be reused for multiple rounds without using any harsh elution conditions.